Development and genetics of red coloration in the zebrafish relative Danio albolineatus
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Evaluation Summary:
Huang et al. address an intriguing question of pigment cell diversification in vertebrates, namely what is the relationship between (yellow) xanthophores and (red) erythrophores. Their data point to a very close relationship between the two cell types, consistent with the view that they are similar cell types differing principally in the details of their pigment biochemistry. The paper will be of interest to scientists across across a range of the many disciplines within pigmentary biology, including developmental biologists, evolutionary biologists, and those who study the chemistry of pigmentation.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Abstract
Animal pigment patterns play important roles in behavior and, in many species, red coloration serves as an honest signal of individual quality in mate choice. Among Danio fishes, some species develop erythrophores, pigment cells that contain red ketocarotenoids, whereas other species, like zebrafish ( D. rerio ) only have yellow xanthophores. Here, we use pearl danio ( D. albolineatus ) to assess the developmental origin of erythrophores and their mechanisms of differentiation. We show that erythrophores in the fin of D. albolineatus share a common progenitor with xanthophores and maintain plasticity in cell fate even after differentiation. We further identify the predominant ketocarotenoids that confer red coloration to erythrophores and use reverse genetics to pinpoint genes required for the differentiation and maintenance of these cells. Our analyses are a first step toward defining the mechanisms underlying the development of erythrophore-mediated red coloration in Danio and reveal striking parallels with the mechanism of red coloration in birds.
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Author Response:
Reviewer #1:
The authors address an interesting but neglected issue in pigment cell biology, concerning the developmental origin of red erythrophores, especially in relationship to yellow xanthophores, and the genetic basis for their differing pigmentation. Red-yellow colouration in vertebrates usually arises from accumulation of dietary carotenoids, and often has significant behavioural importance, e.g. as an honest signal of individual quality. This and the biochemistry of carotenoid colour variation is nicely covered in the Introduction, providing helpful background to a broad audience.
The authors document the widespread presence of erythrophore in Danio, highlighting the unusual nature of Zebrafish within the genus as lacking them. They then develop some quantitative and objective measures of the xanthophores …
Author Response:
Reviewer #1:
The authors address an interesting but neglected issue in pigment cell biology, concerning the developmental origin of red erythrophores, especially in relationship to yellow xanthophores, and the genetic basis for their differing pigmentation. Red-yellow colouration in vertebrates usually arises from accumulation of dietary carotenoids, and often has significant behavioural importance, e.g. as an honest signal of individual quality. This and the biochemistry of carotenoid colour variation is nicely covered in the Introduction, providing helpful background to a broad audience.
The authors document the widespread presence of erythrophore in Danio, highlighting the unusual nature of Zebrafish within the genus as lacking them. They then develop some quantitative and objective measures of the xanthophores and erythrophores based upon Hue and Red:Green autofluorescence ratios, allowing clear distinction of the mature cell-types, and note the often binucleate nature of the erythrophores.
The authors then use a variety of tools to assess, with differing degrees of certainty, the lineage relationships of the erythrophores; together these provide a consistent and convincing picture of shared lineage between the two cell-types. This is consistent with the observed gradual shift in properties of proximal cells from xanthophore-like to erythrophore. A more direct test of the conversion of early xanthophores to erythrophores comes from the clonal analysis of aox5:nucEosFP cells (Fig. 4). They then use a fin regeneration assay to assess the plasticity of these cells in the mature adult. This is a neat experiment, but I am struggling with the interpretation of Figure 5A: which cells are being used as landmarks to justify the conclusion that the cells shown are clonally-derived form that single cell in the 5 dpa image? It may be that the full series of images could be provided in a supplementary figure and might make this clear, but the current images do not seem convincing to me. The experiment in Fig. 5B is convincing, so conclusion seems sound.
We added a supplementary figure (Figure 5—figure supplement 1) to show more context and nearby landmarks, including the amputation plane. We additionally swapped out the images in Figure 5A with an example that more clearly makes our point that cells seem to both lose red coloration and increase in number. Cells of both the original and the new example are visible in the new supplemental figure. Given the concern expressed we additionally modified the salient portion of the text, to make it clearer that the brightfield-only analyses were intended merely to see if a transformation is plausible, based on overt cell colors and behaviors in the absence of formal clonal analysis. The revised text reads:
“We first assessed the possibility that transfating occurs by repeatedly imaging individual fish in brightfield, to learn whether cells near the amputation plane might lose their red color during regenerate outgrowth. Individual erythrophores could often be reidentified using other cells as well as distinctive features of fin ray bones and joints as landmarks (Figure 5A; Fig- ure 5—figure supplement 1). As regeneration proceeded, small groups of cells having paler red or orange coloration, were sometimes observable where individual cells of deep red col- oration had been found, suggestive of proliferation and dilution of pre-existing pigments. Later, only yellow cells were found in these same locations. These observations were con- sistent with the possibility of erythrophore → xanthophore conversion, and so to test this idea directly we marked nucEosFP+ erythrophores by photoconversion prior to amputation (Figure 5B; Figure 5—figure supplement 2A). ”
The authors then use a transcriptomic comparison to identify candidate genes influencing erythrophore v xanthophore differentiation. They study 3 with mutant phenotypes affecting these cell-types, identifying likely roles of 3 erythrophore genes. Whilst most of this analysis is beautifully presented, I am confused by Fig. 7 in which I think panel D and F as described in the legend are inverted.
We fixed the relative ordering of panels and legends. We also changed the Y axis label in Figure 7F to indicate cells per 40 μm2 rather than density, which might be misinterpreted to mean cells per mm.
As is expected form this lab, the manuscript is generally very carefully and clearly written and includes thorough data presentation and statistical analysis. Conclusions drawn are appropriately nuanced, and justified by data presented. The manuscript provides an important first step in understanding the developmental relationship of erythrophores to xanthophores, and a number of genetic resources for the further exploration of this question.
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Evaluation Summary:
Huang et al. address an intriguing question of pigment cell diversification in vertebrates, namely what is the relationship between (yellow) xanthophores and (red) erythrophores. Their data point to a very close relationship between the two cell types, consistent with the view that they are similar cell types differing principally in the details of their pigment biochemistry. The paper will be of interest to scientists across across a range of the many disciplines within pigmentary biology, including developmental biologists, evolutionary biologists, and those who study the chemistry of pigmentation.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name …
Evaluation Summary:
Huang et al. address an intriguing question of pigment cell diversification in vertebrates, namely what is the relationship between (yellow) xanthophores and (red) erythrophores. Their data point to a very close relationship between the two cell types, consistent with the view that they are similar cell types differing principally in the details of their pigment biochemistry. The paper will be of interest to scientists across across a range of the many disciplines within pigmentary biology, including developmental biologists, evolutionary biologists, and those who study the chemistry of pigmentation.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
The authors address an interesting but neglected issue in pigment cell biology, concerning the developmental origin of red erythrophores, especially in relationship to yellow xanthophores, and the genetic basis for their differing pigmentation. Red-yellow colouration in vertebrates usually arises from accumulation of dietary carotenoids, and often has significant behavioural importance, e.g. as an honest signal of individual quality. This and the biochemistry of carotenoid colour variation is nicely covered in the Introduction, providing helpful background to a broad audience.
The authors document the widespread presence of erythrophore in Danio, highlighting the unusual nature of Zebrafish within the genus as lacking them. They then develop some quantitative and objective measures of the xanthophores and …
Reviewer #1 (Public Review):
The authors address an interesting but neglected issue in pigment cell biology, concerning the developmental origin of red erythrophores, especially in relationship to yellow xanthophores, and the genetic basis for their differing pigmentation. Red-yellow colouration in vertebrates usually arises from accumulation of dietary carotenoids, and often has significant behavioural importance, e.g. as an honest signal of individual quality. This and the biochemistry of carotenoid colour variation is nicely covered in the Introduction, providing helpful background to a broad audience.
The authors document the widespread presence of erythrophore in Danio, highlighting the unusual nature of Zebrafish within the genus as lacking them. They then develop some quantitative and objective measures of the xanthophores and erythrophores based upon Hue and Red:Green autofluorescence ratios, allowing clear distinction of the mature cell-types, and note the often binucleate nature of the erythrophores.
The authors then use a variety of tools to assess, with differing degrees of certainty, the lineage relationships of the erythrophores; together these provide a consistent and convincing picture of shared lineage between the two cell-types. This is consistent with the observed gradual shift in properties of proximal cells from xanthophore-like to erythrophore. A more direct test of the conversion of early xanthophores to erythrophores comes from the clonal analysis of aox5:nucEosFP cells (Fig. 4). They then use a fin regeneration assay to assess the plasticity of these cells in the mature adult. This is a neat experiment, but I am struggling with the interpretation of Figure 5A: which cells are being used as landmarks to justify the conclusion that the cells shown are clonally-derived form that single cell in the 5 dpa image? It may be that the full series of images could be provided in a supplementary figure and might make this clear, but the current images do not seem convincing to me. The experiment in Fig. 5B is convincing, so conclusion seems sound.
The authors then use a transcriptomic comparison to identify candidate genes influencing erythrophore v xanthophore differentiation. They study 3 with mutant phenotypes affecting these cell-types, identifying likely roles of 3 erythrophore genes. Whilst most of this analysis is beautifully presented, I am confused by Fig. 7 in which I think panel D and F as described in the legend are inverted.
As is expected form this lab, the manuscript is generally very carefully and clearly written and includes thorough data presentation and statistical analysis. Conclusions drawn are appropriately nuanced, and justified by data presented. The manuscript provides an important first step in understanding the developmental relationship of erythrophores to xanthophores, and a number of genetic resources for the further exploration of this question.
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Reviewer #2 (Public Review):
In this paper the authors present a number of approaches to address the biology of the erythrophore, a red-pigmented cell present in non-mammalian vertebrates including birds, fishes and amphibians. This cell type is found in many species of the genus Danio but not in the widely-studied zebrafish (Danio rerio), therefore the investigators undertake studies in the relative Danio albolineatus, which has a prominent population of erythrophores in its proximal anal fin, with xanthophores (yellow-pigmented cells) located more distally on the anal fin. Both cell types use carotenoid derivatives as pigments, and this spatial arrangement provides an opportunity to address hypotheses concerning their ontogenetic relationship. The authors initially provide two lines of evidence that erythrophores and xanthophores …
Reviewer #2 (Public Review):
In this paper the authors present a number of approaches to address the biology of the erythrophore, a red-pigmented cell present in non-mammalian vertebrates including birds, fishes and amphibians. This cell type is found in many species of the genus Danio but not in the widely-studied zebrafish (Danio rerio), therefore the investigators undertake studies in the relative Danio albolineatus, which has a prominent population of erythrophores in its proximal anal fin, with xanthophores (yellow-pigmented cells) located more distally on the anal fin. Both cell types use carotenoid derivatives as pigments, and this spatial arrangement provides an opportunity to address hypotheses concerning their ontogenetic relationship. The authors initially provide two lines of evidence that erythrophores and xanthophores arise from a common progenitor: fate-mapping via mosaic labeling of cells with transgenic markers as well as a stable line expressing a photoconvertible FP, and analysis of residual clones of pigmented cells in fish targeted by CRISPR to eliminate the scarb1 gene required for carotenoid uptake. Additionally, these experiments suggest that both cell types arise in the fin via differentiation from an unpigmented progenitor, as opposed to migration of committed or differentiatied cells from the body of the fish. The presence of bipotential progenitors is shown to be restricted to cells in the distal portion of the fin, while examination of regrowth of amputated fins reveals that xanthophores can be regenerated from unpigmented precursors as well as from division and transdifferentiation of erythrophores from the proximal fin "stub". In the last half of the paper, the authors identify a set of differentially-expressed genes between the erythrophore-enriched and xanthophore-enriched regions of the anal fin and subject a number of those from the former to CRISPR mutagenesis, with notable hits in the cyp2ae2 and bdh1a loci diminishing red coloration (and as shown by HPLC, levels of the corresponding ketocarotenoid astaxanthin.) The result with cyp2ae2 is especially intriguing considering the association of a related cyp2 P450 gene with red coloration in avians. The authors' conclusions are well-supported by their use of complementary approaches and clear presentation of a wide range of types of data.
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